Fig. 2.
Mimicking phospho-Ser25 protects from TNF-induced cell death. a–g Ripk1+/+ and Ripk1S25D/S25D BMDMs (a, d) or MEFs (b, c, e–g) were pretreated with the indicated compounds for 30 min before stimulation with hTNF (1 ng/ml for BMDMs (a, d) and 100 pg/ml (b, e) or 20 ng/ml (c, f, g) for MEFs). Activation of cytosolic proteins was monitored by immunoblotting (c, f, g) and cell death was measured in function of time by SytoxGreen positivity (a, b, d, e). Cell death data are presented as mean ± SEM of three independent experiments (n = 3). BMDMs results were obtained with cells isolated from three different mice for each genotype (n = 3) (a, d). Statistical significance for the cell death assays was determined using two-way ANOVA followed by a Tukey post-hoc test. h In order to demonstrate efficacy of the IKKi when administered in vivo, mice were i.v. injected with IKKi (200 µg/20 g) 15 min prior to i.p. injection of mTNF (1 µg/20 g) in WT mice. Liver lysates were prepared and protein levels were determined by immunoblot. i–j Ripk1+/+, Ripk1S25D/+, and Ripk1S25D/S25D mice were i.v. injected with IKKi (200 µg/20 g) 15 min prior to i.v. injection of mTNF (150 ng/20 g). k–l Ripk1+/+, Ripk1S25D/+, and Ripk1S25D/S25D mice were i.v. injected with mTNF (10 µg/20 g). Cumulative survival rates (i, k) and body temperature (j, l) were determined in function of time. The number of mice (n) used in each condition is indicated. The temperature results are represented as mean ± SEM. Statistical significance for body temperatures of the mice were determined using two-way ANOVA followed by a Tukey post-hoc test. Survival curves were compared using log-rank Mantel–Cox test. Significance between samples is indicated in the figures as follows: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns non-significant. Immunoblots are representative of 2 (c, f, g–h) independent experiments